Cloning and Construction of N.otophora Chloroplast...

Cloning and Construction of N.otophora Chloroplast Genome Library

The restriction enzyme BamHI was used to construct a genome library of N.otophora ct-DNA because it has a single site within the tetracycline resistance gene of the plasmid pBR322（Bolviar et al.，1977）.BamHI generated 28 fragments of N.otophora ct-DNA.In comparison，SmaI generated fewer fragments and EcoRI generated more fragments（see Figure 1）.

Initially，a mixture of N.otophora ct-DNA and plasmid pBR322 was digested with BamHI，ligated and introduced into E.coli（see materials and methods）.The transformed E.coli cells were first selected on ampicillin plates and then screened for tetracycline sensitivity by dual plating on ampicillin and on tetracycline plates.The frequency of transformation is about 0.5%.Moreover，the frequency of insertion is much higher，a total of 207 recombinants（Ampr，Tcs）were obtained from 2002 colonies（Ampr）.After digestion with BamHI，the chimeric plasmids carrying ct-DNA inserts of various sizes in the recombinants were further analyzed electrophoretically and compared with plasmid pBR322 and BamHI treated N.otophora ct-DNA.Most of the BamHI ct-DNA fragments（23/28）have been cloned in E.coli HB101.Plasmid pBR322 can carry up to four different BamHI ct-DNA fragments.The largest ct-DNA fragment inserted is twice the size of plasmid pBR322.Fragments larger than this，such as BamHI ct-DNA fragments 1，2 and 3，seem to encounter difficulty in being inserted into plasmid pBR322.Therefore those fragments have not been successfully cloned by using the shot gun method with plasmid pBR322 as vehicle in this manner.Alternatively，the BamHI fragments 1，2，3 of N.otophora ct-DNA were separated and recovered from the low-gelling-temperature-agarose gel，then ligated to pBR322 and introduced into E.coli individually.Thus，except BamHI fragment 8，a clone library of the entire N.otophora chloroplast genome has been constructed（see Figure 2）.(https://www.daowen.com)